Background: Adult T-cell leukemia/lymphoma (ATL) is an aggressive malignancy of CD4+CD25+ lymphocytes caused by human T-cell lymphotropic virus type 1. While much progress has been made in understanding the mechanisms of cellular dysregulation, the prognosis for aggressive ATL still remains poor. Therefore, new therapeutic approaches need to be developed.

Results: Previously, we demonstrated that the viral protein Tax inactivates p53 in HTLV-1-infected T-cells. Here we show that 9-aminoacridine (9AA) through p53 reactivation and NF-κB inhibition has selective toxicity for infected leukemic cells independent of their p53 status. We further demonstrate that 9AA activates caspase-3/7 resulting in PARP cleavage. Next we investigated the efficacy of 9AA in the MET-1 ATL model. Alone, 9AA did not cause significant drops in surrogate tumor markers, soluble IL-2Rα or β2-micorglobulin (β2μ) levels with only a slight increase in survival of MET-1-bearing mice. However, in combination with Campath-1H, 9AA treatment resulted in low soluble IL-2Rα and β2μ levels at 2 and 4 weeks. Consistent with reduced tumor cell burden, combination treatment significantly increased survival of MET-1-bearing mice compared to mice treated with either drug alone. Splenic cells isolated from 9AA or combination treated mice showed increased p53 protein levels and transcriptional activity. Consistent with increased tumor suppressor activity, we found increased PARP-1 cleavage in 9AA and combination treated cells.

Conclusion: Our results indicate that targeting reactivation of p53 and inhibition of NF-κB with acridine-derivatives in combination with other chemotherapeutics could result in increased efficacy and selective killing of tumor cells.

Figure 5: 9AA combined with Campath-1H significantly prolonged survival of MET-1 leukemia-bearing mice. (A) On day 1, the serum levels of sIL-2Rα for the four groups ranged from 3,189 to 3,486 pg/mL. Two weeks after therapy, the serum values rose to 72,737 pg/mL for control mice and 32, 654 pg/ml for 9AA mice (9AA vs control, p > .05). The sIL-2Rα levels for Campath-1H (Campath-1) treated mice showed no increase compared to initial levels, 3,270 pg/mL (Campath-1H vs control, p < .01). The combination group decreased to 1,810 pg/mL (combination vs control, or vs 9AA, p < .01). Four weeks after therapy, sIL-2Rα was 279,302 pg/mL and 102,233 pg/mL for control and 9AA groups, respectively (p < .01). Serum sIL-2Rα for the Campath-1H group increased to 7,674 pg/mL (Campath-1H vs control, p < .001). The combination group remained at 1,330 pg/mL (combination vs control, or vs 9AA, p < .001). (B) On day 1, the serum levels of β2μ for the four groups were less than 0.05 μg/mL. Four weeks after therapy, the serum β2μ values of the control and 9AA groups were 9.25 μg/mL and 5.0 μg/mL, respectively (p > .05). The serum β2μ values of the Campath-1H group increased to 0.13 μg/mL (Campath-1H vs control, p < .0001). The serum β2μ values were below detection for the combination group (combination vs control, or vs 9AA, p < .0001). (C) Kaplan-Meier analysis demonstrating combination therapy of 9AA and Campath-1H prolonged survival of MET-1 leukemia-bearing mice. (p < .0001). (D) Four of 8 mice in the Campath-1H group and 8 of 8 mice in the combination group survived to 100 days. The sIL-2Rα levels for the groups of Campath-1H and the combination were 296,467 pg/mL and 4,609 pg/mL, respectively (p < .001).

Mentions:
A 2-week course of treatment with 9AA (150 μg/mouse/day, a 14-day infusion), a 4-week course of treatment with Campath-1H (100 μg/mouse, weekly), and the combination of 9AA and Campath-1H manifested therapeutic efficacy as assessed by demonstration both of reduced serum levels of sIL-2Rα and β2μ and prolongation of survival of the leukemia-bearing mice (Figure 5). When compared with the PEG300 control group of mice, at week 2 and week 4, there was a significant reduction of serum levels of sIL-2Rα in the groups treated with Campath-1H alone (p < .01), and the combination of 9AA with Campath-1H (p < .01, Figure 5A) as well as a reduction in serum β2μ levels at week 2 and week 4 in the Campath-1H alone group (p < .0001) and in the combination group ( p < .0001, Figure 5B). Furthermore, the mean sIL-2Rα levels were significantly lower in the combination group 100 days post-therapy when compared with that of the group that received Campath-1H alone (p < .001, Figure 5D).

Figure 5: 9AA combined with Campath-1H significantly prolonged survival of MET-1 leukemia-bearing mice. (A) On day 1, the serum levels of sIL-2Rα for the four groups ranged from 3,189 to 3,486 pg/mL. Two weeks after therapy, the serum values rose to 72,737 pg/mL for control mice and 32, 654 pg/ml for 9AA mice (9AA vs control, p > .05). The sIL-2Rα levels for Campath-1H (Campath-1) treated mice showed no increase compared to initial levels, 3,270 pg/mL (Campath-1H vs control, p < .01). The combination group decreased to 1,810 pg/mL (combination vs control, or vs 9AA, p < .01). Four weeks after therapy, sIL-2Rα was 279,302 pg/mL and 102,233 pg/mL for control and 9AA groups, respectively (p < .01). Serum sIL-2Rα for the Campath-1H group increased to 7,674 pg/mL (Campath-1H vs control, p < .001). The combination group remained at 1,330 pg/mL (combination vs control, or vs 9AA, p < .001). (B) On day 1, the serum levels of β2μ for the four groups were less than 0.05 μg/mL. Four weeks after therapy, the serum β2μ values of the control and 9AA groups were 9.25 μg/mL and 5.0 μg/mL, respectively (p > .05). The serum β2μ values of the Campath-1H group increased to 0.13 μg/mL (Campath-1H vs control, p < .0001). The serum β2μ values were below detection for the combination group (combination vs control, or vs 9AA, p < .0001). (C) Kaplan-Meier analysis demonstrating combination therapy of 9AA and Campath-1H prolonged survival of MET-1 leukemia-bearing mice. (p < .0001). (D) Four of 8 mice in the Campath-1H group and 8 of 8 mice in the combination group survived to 100 days. The sIL-2Rα levels for the groups of Campath-1H and the combination were 296,467 pg/mL and 4,609 pg/mL, respectively (p < .001).

Mentions:
A 2-week course of treatment with 9AA (150 μg/mouse/day, a 14-day infusion), a 4-week course of treatment with Campath-1H (100 μg/mouse, weekly), and the combination of 9AA and Campath-1H manifested therapeutic efficacy as assessed by demonstration both of reduced serum levels of sIL-2Rα and β2μ and prolongation of survival of the leukemia-bearing mice (Figure 5). When compared with the PEG300 control group of mice, at week 2 and week 4, there was a significant reduction of serum levels of sIL-2Rα in the groups treated with Campath-1H alone (p < .01), and the combination of 9AA with Campath-1H (p < .01, Figure 5A) as well as a reduction in serum β2μ levels at week 2 and week 4 in the Campath-1H alone group (p < .0001) and in the combination group ( p < .0001, Figure 5B). Furthermore, the mean sIL-2Rα levels were significantly lower in the combination group 100 days post-therapy when compared with that of the group that received Campath-1H alone (p < .001, Figure 5D).

Background: Adult T-cell leukemia/lymphoma (ATL) is an aggressive malignancy of CD4+CD25+ lymphocytes caused by human T-cell lymphotropic virus type 1. While much progress has been made in understanding the mechanisms of cellular dysregulation, the prognosis for aggressive ATL still remains poor. Therefore, new therapeutic approaches need to be developed.

Results: Previously, we demonstrated that the viral protein Tax inactivates p53 in HTLV-1-infected T-cells. Here we show that 9-aminoacridine (9AA) through p53 reactivation and NF-κB inhibition has selective toxicity for infected leukemic cells independent of their p53 status. We further demonstrate that 9AA activates caspase-3/7 resulting in PARP cleavage. Next we investigated the efficacy of 9AA in the MET-1 ATL model. Alone, 9AA did not cause significant drops in surrogate tumor markers, soluble IL-2Rα or β2-micorglobulin (β2μ) levels with only a slight increase in survival of MET-1-bearing mice. However, in combination with Campath-1H, 9AA treatment resulted in low soluble IL-2Rα and β2μ levels at 2 and 4 weeks. Consistent with reduced tumor cell burden, combination treatment significantly increased survival of MET-1-bearing mice compared to mice treated with either drug alone. Splenic cells isolated from 9AA or combination treated mice showed increased p53 protein levels and transcriptional activity. Consistent with increased tumor suppressor activity, we found increased PARP-1 cleavage in 9AA and combination treated cells.

Conclusion: Our results indicate that targeting reactivation of p53 and inhibition of NF-κB with acridine-derivatives in combination with other chemotherapeutics could result in increased efficacy and selective killing of tumor cells.